The present invention relates to a novel process for labeling a ribonucleic acid (RNA) with signal amplification.
The state of the art shows that there are a large number of methods for labeling nucleotides, oligonucleotides or nucleic acids; oligonucleotides and nucleic acids will be referred to by the term polynucleotides. Polynucleotides can be labeled either during synthesis or by incorporating at least one labeled nucleotide.
A first method comprises in attaching the label to the base, whether the latter is a natural base or a modified base. A second method proposes attaching the label to the sugar, again whether the latter be a natural sugar or a modified sugar. A third method relates to attaching the label to the phosphate.
In fact, a person of skill in the art who is to label a nucleotide or a nucleotide analogue or a nucleic acid is inclined to attach the label to the base or to the sugar, which offers him more convenience and more options. This is, furthermore, what emerges from studying a large number of documents such as EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879, U.S. Pat. Nos. 5,449,767, 5,328,824, WO-A-93/16094, DE-A-3.910.151 and EP-A-0.567.841 in the case of the base or EP-A-0.286.898 in the case of the sugar. Each of these documents is hereby incorporated by reference for all purposes.
The technique of attaching the label to the phosphate is more complex especially because nucleic acids are water soluble and the reactivity of phosphate in this media is lower compared to that in organic solvents.
Even so, some documents have proposed techniques for labeling the phosphate. This applies, for example, to document EP-A-0.280.058, hereby incorporated by reference for all purposes, which describes labeling a nucleotide by attaching the label to the phosphate, with the latter being attached to the sugar in the 3xe2x80x2 and/or 5xe2x80x2 positions, when the nucleotide is a deoxyribonucleotide, and in the 2xe2x80x2, 3xe2x80x2 and/or 5xe2x80x2 positions when the nucleotide is a ribonucleotide. This document also describes a polynucleotide or oligonucleotide which comprises at least one labeled nucleotide as described above; this nucleotide is incorporated into the polynucleotide or oligonucleotide during synthesis.
However, the labeling strategy which is proposed by document EP-A-0.280.058 does not enable the nucleic acids to be labeled uniformly. The incorporation of the labeled nucleotides into the polynucleotides cannot be controlled; it depends entirely on the composition of synthesized polynucleotides. Thus, some polynucleotides may contain a large number of labeled nucleotides whereas others may not contain any at all. As a result, the intensity of the signal emitted by these nucleic acids will not be uniform, and therefore it will be difficult to interpret the results when detecting the nucleic acids.
In this case, the labeling is incorporated biologically without any control of the positions of the labeled nucleotides.
The document U.S. Pat. No. 5,317,098 hereby incorporated by reference for all purposes relates to nucleic acids which are labeled at their 5xe2x80x2 ends. This attachment uses imidazole and a linker arm. There is no associated fragmentation with the labeling. Furthermore, phosphate is added to nucleic acids and therefore kinase is used as a mean to introduce the phosphate, leading to at least one additional biological step. This document describes the labeling of a 15 mer oligonucleotide. When using large nucleic acids instead of oligonucleotide, this technique leads to the presence of a label only at the 5xe2x80x2 end and the specific activity of the labeled nucleic acid is low.
In addition, when the labeling is carried out on large nucleic acids without a fragmentation stage, also termed a cleavage stage, the kinetics of hybridization of these labeled nucleic acids to their complementary sequences, is slow leading to poor hybridization yield. This will therefore result in a quantitative and qualitative loss of the signal. Steric hindrance is a key factor in this reaction.
Steric hindrance may not only be the result of the length of the nucleic acid but also of the existence of secondary structures. Fragmentation helps to broke (or reduce) these structures and in this way to optimize hybridization. Steric hindrance plays a particularly important role in the case of hybridization to solid surfaces which contain a high density of capture probes, for example the DNA arrays developed by the company Affymetrix, Inc. (xe2x80x9cAccessing Genetic Information with High-Density DNA arraysxe2x80x9d, M. Chee et al., Science, 274, 610-614, 1996. xe2x80x9cLight-generated oligonucleotide arrays for rapid DNA sequence analysisxe2x80x9d, A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 91, 5022-5026, 1994, U.S. Pat. Nos. 5,744,305, 5,445,934). Each of these references is incorporated therein by reference for all purposes. In this technology, the capture probes are generally of reduced size, being of about twenty nucleotides in length.
A large number of methods are described in the state of the art for fragmenting nucleic acids.
First, the fragmentation can be enzymatic, i.e. the nucleic acids can be fragmented by nucleases (DNases or RNases) (Methods in Enzymol., vol. 152, S. Berger and A. Kimmel, ed. Academic Press, 1987, Enzymatic techniques and Recombinant DNA Technology ,  less than Guide to Molecular cloning  greater than , p91-110, Molecular Cloning, a Laboratory Manual, J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, 2nd Edition, p5.30-5.95, 1989). Each of these documents is hereby incorporated by reference for all purposes. Depending on the involved enzyme, this reaction generates small fragments or monomers having either a hydroxyl or a monophosphate group at their 5xe2x80x2-or 3xe2x80x2-ends.
Second, the fragmentation can be chemical. For example, in the case of DNA sequences, the depurination or depyrimidination using alkylating agents generates abasic sites which are then fragmented in the presence of a base by a mechanism termed xe2x80x9cxcex2-eliminationxe2x80x9d (T. Lindahl et al., Rate of Chain breakage at apurinic sites in double-stranded deoxyribonucleic acid., Biochemistry, 11, p3618-3623, 1972). The DNA""s can be fragmented by oxidation, alkylation or free radical addition mechanisms, inter alia (M. Liuzzi et al., Characterization and damage in gamma-irradiated and OsO4-treated DNA using methoxyamine., Int. J. Radiat. Biol., 54, p709-722, 1988). Metal cations, which are often combined with organic molecules used as chemical catalysts, for example imidazole, are used for fragmenting RNA""s. (R. Breslow and R. Xu, Recognition and catalysis in nucleic acid chemistry, Proc. Natl. Acad. Sci. USA, 90, p1201-1207, 1993. J. Hovinen et al. Imidazole Tethered oligonucleotides: Synthesis and R cleaving activity, J. Org. Chem., 60, p2205-2209, 1995). This fragmentation is preferably carried out in an alkaline medium and generates fragments having 3xe2x80x2-phosphate ends. Each of these documents is hereby incorporated by reference for all purposes.
However, the objective of these fragmentations is not that of facilitating or permitting labeling.
Document WO-A-88/04300 proposes a method for fragmenting and labeling RNA, using RNA molecules which possesses enzymatic properties, i.e. ribozymes. Cleavage catalysis with these ribozymes is sequence specific and the reaction yields to RNA fragments having a hydroxyl group (OH) at their 5xe2x80x2 end and a monophosphate at 3xe2x80x2 end. The labeling, which is solely radioactive labeling, is then effected by incorporating an added radioactive phosphate which is derived from a molecule of GTP. It is a phosphotransferase activity of these ribozymes category, i.e a kinase activity. The radioactive phosphate attachment is effected solely at the hydroxyl group at 5xe2x80x2 end and no phosphate resulting from fragmentation is used for attaching the label to RNA fragments. Furthermore, the fragmentation is only carried out by ribozymes, implying the existence of a specificity between the ribozymes and the target nucleic acids to be cleaved. The phosphate then acts as the label.
Our invention allows the label attachment to the phosphate of a nucleic acid fragment which is released during the cleavage. There is no specificity and therefore, any type of nucleic acid can be fragmented in a random manner. The homogeneity of labeling intensity can be obtained using this approach since the labeling yield of each class of produced fragments is completely independent on its sequence and composition. Thus, our process makes it possible to prepare detection probes, for example. Finally, the phosphate is only a linker arm between the nucleic acid and the label.
The technique of signal amplification is well known in the field of immunoassays or nucleic acid probes as described for example in WO95/08000, or in the article J. Histochem. Cytochem. 45: 481-491, 1997 (each of which is incorporated by reference in its entirety for all purposes), but without fragmentation associated during the labeling.
No process of fragmenting before labeling with signal amplification has been described in the prior art.
The present invention therefore proposes a process which overcomes the previously mentioned drawbacks. Thus, this process makes it possible to obtain RNA fragments which are uniformly labeled once the fragmentation has been completed. In addition, the fragmentation makes it possible to obtain fragments which are of an optimum size for a possible hybridization. With the quality of the hybridization having been improved, the post-hybridization detection of labeled fragments will be more rapid and efficient. Finally, the invention improves the sensitivity by increasing the signal intensity produced and the ratio signal versus the background.
To this end, the present invention relates to a process for labeling a synthetic or natural ribonucleic acid (RNA), characterized in that it comprises:
fragmenting the RNA,
fixing a first ligand to the terminal phosphate which is located at the 3xe2x80x2 end and/or the 5xe2x80x2 end of each fragment of said RNA, said terminal phosphate having been released during the fragmentation, and
binding a labeling agent to said first ligand.
In the present invention, RNA (ribonucleic acid or polyribonucleic acid) is a synthetic or natural RNA.
Those of skill in the art will be familiar with methods of obtaining synthetic RNA. These methods include, for example, amplification techniques (see, for example, Kozal M. J. and al, Nature Medicine, 2(7), 753-758, 1996, hereby incorporated by reference in its entirety for all purposes), transcriptional amplification techniques or other methods leading to RNA products including TMA (Transcription Mediated Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), 3SR (Self-Sustained Sequence Amplification), Qxcex2 replicase amplification, natural RNA digestion by enzymes, and polyribonucleotide chemical synthesis (see, for example, U.S. Pat. Nos. 5,554,516 and 5,766,849 and Clin. Microbial. Rev.,5(4), p.370-386, 1992 each of which is incorporated by reference in its entirety for all purposes. Synthetic RNA is also RNA which comprises at least one modified nucleotide or at least one modified internucleotidic bond such as thiophosphate. A Natural RNA is a RNA which is obtained by extraction from a cell, for example a messenger RNA (mRNA), a ribosomal RNA (rRNA) or a transfer RNA (tRNA). Labeling is the attachment of a label which is able to generate a detectable signal. The following is a non-limiting list of these labels:
enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, xcex2-galactosidase and glucose-6-phosphate dehydro-genase,
chromophores, such as fluorescent and luminescent compounds and dyes,
groups having an electron density which can be detected by electron microscopy or by their electrical properties such as conductivity, amperometry, voltametry and impedance,
detectable groups, for example whose molecules are of sizes which are sufficient to induce detectable modifications in their physical and/or chemical characteristics; this detection can be effected by means of optical methods such as diffraction, surface plasmon resonance, surface variation and angle of contact variation, or physical methods such as atomic force spectroscopy and the tunnel effect,
radioactive molecules such as 32P, 35S or 125I.
The compound which comprises the label is the labeling agent.
The term  less than fixing greater than  means creating a covalent or a non covalent bond. An antibody selective of a phosphate or thiophosphate is a means to create a non covalent bond. According to a preferred mode of operation, the fixation is covalent as described in the examples.
In one aspect of the present invention, the fragmentation and the fixation are effected in one step.
In another aspect of the present invention, the fragmentation and the fixation are effected in two steps.
According to a first embodiment, the binding of the labeling agent to the first ligand is covalent. The different reactive functions which allows the covalent coupling are well known to those of skill in the art and some examples of conjugation could be found for example in  less than Bioconjugate techniques greater than , Hermanson G. T., Academic Press, San Diego, 1996. Hereby incorporated by reference in its entirety for all purposes.
According to a second embodiment, the binding of the labeling agent to the first ligand is non covalent. The non covalent binding is a binding involving, for examples, ionic or electrostatic interactions, Van der Vaals"" interactions, hydrogen bonds or a combination of different interactions.
In a preferred embodiment, the binding of the labeling agent to said first ligand is effected indirectly. The first ligand, fixed to the terminal phosphate, is bound to a first antiligand, said first antiligand is bound to a second ligand and the labeling agent is a second antiligand bearing at least one label and able to react with said second ligand.
The (antiligand/ligand) combination means two compounds which are able to react together in a specific manner.
First ligand/first antiligand and second ligand/second antiligand combinations are selected, for example, from the group consisting of biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin and polynucleotide/complementary polynucleotide.
These different combinations and other combinations are known and described for example in BioMerieux applications WO 96/19729, WO 94/29723, WO 95/08000 which are incorporated herein by reference.
The first and second ligands are the same or different.
In a preferred mode of operation, the first ligand is a derivative of fluorescein and the second ligand is a derivative of biotin.
In another preferred mode of operation, the first ligand is a derivative of biotin and the first antiligand is a derivative of streptavidin.
There is no limitation in stacking-up other entities (ligand/antiligand) to increase signal amplification. For example, a (second ligand/first antiligand) entity binds to the first ligand fixed to the phosphate, then a (third ligand/second antiligand) entity binds to the second ligand and the labeling agent is a third antiligand bearing at least one label and able to react with the third ligand.
The addition of different entities may be effected in at least one step or each entity may be added successively after the fixation of the ligand to the phosphate.
According to a preferred mode of operation, the fixation of the first ligand to the 3xe2x80x2 end of each fragment of the RNA is effected apart from the fragment which constitutes the 3xe2x80x2 and/or 5xe2x80x2 end of the starting RNA. Additionally or alternatively, the fixation of first ligand to the 5xe2x80x2 end of each fragment of the RNA is effected apart from the fragment which constitutes the 5xe2x80x2 end of the starting RNA.
Whatever the embodiment, fixation of the first ligand to the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by reacting a reactive function, which is carried out by the first ligand, to the phosphate which is in the 2xe2x80x2 position, in the 3xe2x80x2 position or in the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position, with respect to the ribose.
Fragmentation and/or the fixation of the first ligand to the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by binding a nucleophilic, electrophilic or halide function which is carried by a ligand to the phosphate in the 2xe2x80x2 position, in the 3xe2x80x2 position or in the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position, with respect to the ribose.
Fragmentation of the RNA is effected enzymatically, chemically or physically.
Enzymatic fragmentation of the RNA is carried out by nucleases.
Chemical fragmentation of the RNA is carried out by metal cations which may or may not be combined with a chemical catalyst.
In this case, the metal cations are Mg++, Mn++, Cu++, Co++ and/or Zn++ ions and the chemical catalyst comprises imidazole, a substituted analogue, for example N-methylimidazole, or any chemical molecule which has an affinity for the RNA and which carries an imidazole ring or a substituted analogue.
Physical fragmentation of the RNA is carried out by sonication or by radiation.
In all the cases in point, the fixation of the first ligand to the 3xe2x80x2 end or the 5xe2x80x2 end of an RNA fragment is effected by reacting a molecule R-X, where R comprises the ligand and X is the reactive function, such as a hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide or maleimide. X reacts to the phosphate which is linked to the 2xe2x80x2 position, to the 3xe2x80x2 position or to the cyclic monophosphate 2xe2x80x2-3xe2x80x2 position of the ribose. In a preferred embodiment, X is an alkyl halide, phenylmethyl halide, iodoacetamide or maleimide. In order to facilitate the fixation of the ligand, a linker arm is optionally present between the ligand and the reactive function. In a preferred embodiment, R-X is N-(biotinoyl)-Nxe2x80x2-(iodoacetyl)ethylenediamine, (+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine, N-Iodoacetyl-N-biotinylhexylenediamine, 5-(bromomethyl) fluorescein.
The present invention also comprises a RNA fragment which is obtained by the above process,. The RNA fragment possesses at the 3xe2x80x2 end or the 5xe2x80x2 end a single nucleotide which is labeled at the terminal phosphate which was released during fragmentation.
This RNA fragment comprises from 10 to 150 nucleotides, preferably from 30 to 70 nucleotides and preferably from 40 to 60 nucleotides to facilitate hybridization of the RNA fragment to a probe or a target.
According to a preferred embodiment, the RNA fragment comprises at least one thiophosphate nucleotide.
In addition, the nucleotide bearing the ligand is a thiophosphate nucleotide.
According to a preferred embodiment the RNA fragment comprises at the 3xe2x80x2 end a phosphate or a thiophosphate bearing a fluorescein bound to an anti-fluorescein antibody bearing at least one biotin, said antibody bound to a labeled streptavidin.
The invention relates to the use of an RNA fragment, as defined above, as a probe for detecting an RNA and/or a DNA or an RNA fragment and/or a DNA fragment.
The invention finally relates to the use of an RNA fragment, as defined above, as a labeled target which is able to bind to a capture probe.